Name: mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis
Uploaded: 2015-03-31T15:00:00
Description: Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

We appreciate Dr. Jeong-Soo Lee for sharing the Tg(dbh:EGFP-MYCN) transgenic fish with us in our study. This work was supported by a grant 1K99CA178189-01 from the National Cancer Institute, a fellowship from the Pablove Foundation and the Friends for Life, and young investigator awards from the Alex's Lemonade Stand Foundation and the CureSearch for Children's Cancer Foundation.

注：所有斑马鱼的研究和维护动物都符合梅奥诊所研究所IACUC批准协议＃A41213完成。 1. DNA构建的转基因<ol><li>扩增使用CH211-270H11 BAC克隆（从BACPAC资源中心（BPRC））作为DNA模板，一个5.2-kb的多巴胺β羟化酶（四βH）启动子区8。使用PCR体系适合于长DNA模板长和准确的PCR扩增和随后循环方案进行PCR：2分钟94℃，10个循环的（94℃，15秒，50℃，30秒，68° ℃，8?...

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在这种代表性的研究中，我们采用瞬时共注射和激活ALK的共表达与mCherry报告基因MYCN -expressing转基因鱼，以表明这些基因合作，显着加速神经母细胞瘤发病的，与我们之前的发现，复合稳定转基因鱼共表达一致无论激活ALK和MYCN 8。这种转基因拼接方法具有几个明显的优势比传统方法。最重要的，它使得能够共表达在主要注射的动物的候选癌基因（F0代）而?...

巨蟹座的标志是病理突变，缺失和染色体的收益随着时间的积累渐进的疾病。这些遗传异常可影响多个细胞过程，从细胞周期，细胞死亡，有活力的细胞骨架强调反应，如缺氧的代谢和组装。因此，肿瘤发生反映多种遗传畸变的跨生物过程的频谱集体行动。最近综合基因组的研究工作，包括全基因组测序，基因组测序，测序的目标，深度测序和全基因组关联研究，已确定在几乎所有类型的肿瘤1-4的越来越多的新的遗传变化。在许多情况下，遗传发生病变一起以非随机方式5-8，表明它们在疾病的发病机制的合作。剖析大阵造成˚F异常表达的基因的致癌作用ROM这些基因组的病变有必要设计新的治疗策略，并了解肿瘤细胞对这些药物的反应，但是这已经被证明是一项艰巨的任务，需要非常健壮的动物模型系统对于高通量功能基因组学分析的情况下进行体内 。 虽然哺乳动物，尤其是啮齿动物，青睐车型在癌症生物学，斑马鱼已开始引起相当大的关注。该硬骨斑马鱼（ 达里奥鱼 ）已被用来作为模式生物自20世纪60年代开发研究，并首先应用于肿瘤的发病机制的研究，在1982年9-11。易于维护，小的车身尺寸，以及高繁殖力使得斑马鱼稳健的模型，进行大规模的正向遗传学的屏幕，以识别对异常和病理表现型10的突变。斑马鱼胚胎的光学透明度是支持更广泛地使用这种癌症模型中，作为另一重要特征它允许体内成像的情况下进行，以找到肿瘤的发展实时9，一种应用程序，它是在啮齿动物12相对困难。最近比较基因组学的斑马鱼参考基因组（Zv9）的分析显示26206个蛋白质编码基因，与具有人直向同源物，其中82％的值与在所述在线孟德尔遗传疾病相关基因在曼（OMIM）数据库13 71％， 14。因此，斑马鱼已经被用于模拟不同类型的人类癌症，包括神经母细胞瘤8中，T细胞急性淋巴细胞白血病（T-ALL）15,16，黑素瘤17,18，尤因氏肉瘤19，横纹肌肉瘤20,21，胰腺癌22，肝细胞癌23和髓系恶性血液病24,25，并已被选定作为癌症模型异种移植研究11,26。 一个稳定的转基因在斑马鱼的方法通常用于研究的增益的功能的基因在正常发育或疾病发病27,28的效果。开发这样的模型（ 图1A），一个注射含有感兴趣由一个组织特异性启动子驱动的为单细胞的野生型胚胎的基因的DNA构建物。注射三至四个月后，当注射的胚胎达到性成熟，它们远交，与野生型鱼以筛选那些表示整合的DNA的构建在其种系，哪些许可证它们作为创始人鱼。许多因素，如拷贝数和转基因的整合位点，影响在稳定的转基因系的转基因的表达。因此，开发一种转基因肿瘤模型中，多个稳定的转基因株系过度单一的基因必须首先生成和筛选表达转基因的可能导致肿瘤的诱导，一个水平线上。但是，如果候选人邻表达ncogene是有毒的生殖细胞，很难通过直接过表达的转基因29，以产生一个稳定的转基因系。因此，这种方法可以是耗时的，具有故障，以产生合适的癌症模型中的高风险。 这里，我们说明根据镶嵌瞬时转基因（ 图1B）的替代策略，比传统的稳定的转基因用于功能基因组研究在体内提供了独特的优势。在这种方法中，一个或多个转基因构建体注射到转基因或野生型胚胎的单细胞阶段。含有转基因的注射DNA构建然后mosaically和随机整合到主注入鱼，导致在个体的鱼30的多个细胞群内混合基因型。此外，多种的DNA共注射构建在单细胞胚胎导致共整合到在随机位点相同的细胞，允许一个TRACE细胞与转基因的表达，并在镶嵌31动物疾病的发病机制研究不同基因之间的相互作用。作为原理的证明，我们暂时下多巴胺β羟化酶（四βH）启动子在野生型鱼和转基因鱼过度MYCN的控制过度mutationally活化ALK（F1174L）配mCherry报告基因在周交感神经系统（PSNS）。 ALK，编码受体酪氨酸激酶，是最常见的突变基因中的高风险神经母细胞瘤5-7,32,33。ALK（F1174L），作为最频繁的和有效的体细胞活化突变之一，是在超过限额MYCN-扩增高危神经母细胞瘤的患者，用MYCN表达协同作用，以加速神经母细胞瘤肿瘤发生在两个稳定的转基因小鼠和转基因斑马鱼的模型8,34,35。马赛克ALK（F1174L）配mCherry在MYCN转基因鱼的瞬态过表达，我们概括在稳定的转基因鱼过量表达既ALK（F1174L）和MYCN观察肿瘤发病的加速度，这表明转基因镶嵌策略可用于快速和有效地评估多个癌基因在肿瘤发生在体内的相对贡献。

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			<td height="21" style="height:21px;width:373px;">Name of Material/ Equipment</td>
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			<td height="42" style="height:42px;">Expand Long Template PCR System&#160;</td>
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			<td height="21" style="height:21px;">pCR-TOPO vector&#160;</td>
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			<td style="width:119px;">451641</td>
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			<td height="25" style="height:25px;">T4 DNA ligase</td>
			<td style="width:171px;">New England Biolabs, MA</td>
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			<td height="21" style="height:21px;">Gateway LR Clonase II enzyme 
			Mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11791-100</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#174; BP Clonase&#174; II enzyme mix</td>
			<td style="width:171px;">Invitrogen, CA</td>
			<td>11789-020</td>
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			<td height="24" style="height:25px;">GC-RICH PCR System&#160;</td>
			<td style="width:171px;">Roche Applied Science, IN</td>
			<td>12 140 306 001</td>
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			<td height="26" style="height:26px;">Meganuclease I-SceI&#160;</td>
			<td style="width:171px;">New England Biolabs, MA</td>
			<td>R0694S</td>
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			<td height="21" style="height:21px;">Nikon SMZ-1500 stereoscopic fluorescence microscope&#160;</td>
			<td style="width:171px;">Nikon, NY</td>
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			<td height="21" style="height:21px;">Nikon digital sight DS-U1 camera</td>
			<td style="width:171px;">Nikon, NY</td>
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mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

调查是否mutationally活化ALK F1174L或野生型ALK的过表达可在神经母细胞瘤诱导MYCN协作，我们过表达或者活化的人ALK或野生型人ALK下的转基因鱼过度MYCN的PSN 对dβH启动子的控制。下列任一构建体，DβH的- ALKF1174L或DβH - ALKWT，被共注射与DβH - mCherry成单细胞的野生型或MYCN转基因胚胎（ 图2A）8。</s...

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis at JoVE.com

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

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Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

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发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...